TG2 INHIBITORS FOR IMPROVING MUCOCILIARY CLEARANCE IN RESPIRATORY DISEASES

Abstract

In asthma, modification of gel-forming respiratory mucins leading to their tethering to the apical pole of epithelial cells, are believed to participate in airway obstruction by mucus plugs. These changes have been linked to local production of Th2 cytokines, resulting in mucus cell hyperplasia and increased MUC5AC production. The inventors showed that severe eosinophil asthma was associated with overexpression of transglutaminase 2 (TG2), an enzyme recently involved in intestinal mucin reticulation. Moreover, the bronchial epithelium from asthmatic patients or control subjects was reconstituted in vitro by culturing cells at the air-liquid interface and the hypersecretory differentiation was modeled by exposing control bronchial epithelial to IL-13. The inventors showed TG2 expression was upregulated upon IL-13-mediated hypersecretory differentiation and correlated with MUC5AC expression. IL-13 promoted MU5AC tethering to in vitro reconstituted hypersecretory epithelium, and this was blocked by a TG2 inhibitor. In conclusion, the inventors showed that TG2 participates in respiratory mucin modifications in asthma, and contribute to mucus tethering to the airway wall, supporting the use of TG2 inhibitors for improving mucociliary clearance in asthma, but more generally in respiratory diseases.

Claims

1. A method of improving mucociliary clearance, increasing the liquefaction of mucus, preventing mucus tethering to the airway wall, preventing extensive airway mucus plugging and/or preventing mucus occlusion of an airway lumen in a patient suffering from a chronic airway disease, comprising administering to the patient a therapeutically effective amount of a TG2 inhibitor.

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. The method according to claim 1, wherein the patient has a chronic airway disease which causes abnormal or excessive viscoelasticity or cohesiveness of mucus.

7. The method according to claim 1, wherein the patient has a chronic airway disease selected from, cystic fibrosis (CF), chronic obstructive pulmonary disease, bronchiectasis and asthma.

8. The method according to claim 1, wherein the patient suffers from asthma.

9. The method according to claim 1, wherein the TG2 inhibitor is a small organic molecule or an antibody.

10. The method according to claim 1, wherein the TG2 inhibitor is an inhibitor of gene expression that is a siRNA, an antisense oligonucleotide or a ribozyme.

11. A method for increasing the lung delivery of nanoparticles in a patient in need thereof comprising administering the nanoparticles in combination with an amount of a TG2 inhibitor.

12. The method of claim 11 wherein the nanoparticles have encapsulated therein, dispersed therein, and/or covalently or non-covalently associated with a surface thereof one or more therapeutic or diagnostic agents.

13. The method of claim 12 wherein the one or more therapeutic agents is selected from the group consisting of analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antipsychotic agents, neuroprotective agents, anti-proliferatives, such as anti-cancer agent, anti-infectious agents, such as antibacterial agents and antifungal agents, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics and anti-narcoleptics.

14. The method of claim 12 wherein the one or more diagnostic agents is selected from the group consisting of paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides.

15. A mucus-penetrating nanoparticle coated with a TG2 inhibitor.

16. The method according to claim 8, wherein the asthma is severe asthma.

Description

FIGURES

[0061] FIG. 1: TG2 expression is upregulated in MUC5AC hyperexpressing epithelia A: TG2 and MUC5AC mRNA were quantified by RT-qPCR in in vitro regenerated bronchial epithelia by culturing cells from controls (n=18) and asthma subjects (n=56) at the air-liquid (ALI) interface. Spearman correlation analysis (r) is shown). B: TG2 mRNA upregulation during IL-13-induced mucus cell hyperplasia (black bars) as compared to control epithelial cell differentiation (white bars) in ALI culture. n=3.

[0062] FIG. 2: TG2 upregulation is observed in in vitro regenerated bronchial epithelia in asthma and is associated with poorer mucociliary clearance A: TG2 mRNA were quantified by RT-qPCR in ALI-regenerated bronchial epithelia from controls (n=18) and asthma subjects (n=56). B: Mean velocity of fluorescent microspheres deposited on ALI-epithelia from controls and TG2-low or TG2-high asthma patients and analysed using a Zeiss videomicroscope and ImageJ (MTrack3 Plugin). Median values are shown. *p<0.05

[0063] FIG. 3: TG2 expression levels are upregulated in ALI-regenerated epithelia from cystic fibrosis and asthma patients and correlate with TGF-beta2 expression levels. TG2 and TGF-beta2 mRNA levels were quantified by microarray (A) or RT-qPCR (B and C) in cystic fibrosis (A and B, n=4) and asthma (C, n=49) patients. A: Data are expressed as mean S.E.M. * p<0.05. B and C: Correlation analysis of TG2 and TGF-beta2 levels (Spearman (r)) in cystic fibrosis (B) and asthma (C) sets.

[0064] FIG. 4: Respiratory mucins are TG2 substrates Respiratory mucins secreted by ALI-regenerated epithelia were reduced, alkylated and subsequently exposed to human recombinant TG2 with a biotinylated amine (BAP) in the absence (line 1) or presence of ZDON (line 3) or of its vehicle (line 2) for 24h @ 37 C. Mucins were then separated using hybrid SDS agarose/polyacrylamide gel, transferred on a PVDF membrane and BAP incorporation was detected using streptavidine-HRP.

[0065] FIG. 5: The TG2 inhibitor ZDON decreases MUC5AC tethering to the apical surface of hypersecretory epithelia MUC5AC was detected by immunofluorescence in ALI-regenerated hypersecretory epithelia after extensive apical washes with PBS in sections (A and B) or in whole-mount preparations (C and D). Whole-mount images represent stacks from optical sections done through the mucus (parallel to the plane of the epithelium). epi, epithelium. Extracellular mucus was quantified after beta-tubulin staining to delineate cell apex, and expressed as sum intensities (in nm 2 or nm 3) (n=2).

[0066] FIG. 6: The TG2 inhibitor ZDON tends to ameliorate mucociliary clearance in hypersecretory epithelia A: Paths of fluorescent microspheres deposited on IL-13-stimulated hypersecretory epithelia. The final image from a 30-second image sequence (every 5 ms) is shown (ImageJ, Z Project). B: Quantification of travelled distance (ImageJ, Plugin MTrack3). Median values are shown. n=2.

EXAMPLE

[0067] Methods: The bronchial epithelium from asthmatic patients, cystic fibrosis or control subjects was reconstituted in vitro by culturing cells at the air-liquid interface. Hyper secretory differentiation was modeled by exposing control bronchial epithelial to IL-13. Expression of TG2 and MUC5AC was assessed by RT-qPCR and immunohistochemistry/immunofluorescence. Mucin modification by TG2 was evaluated by BAP incorporation and Western-blot analysis. Mucus tethering to the apical surface was studied by detecting MUC5AC after extensive apical washes with PBS. The role of TG2 was evaluated using the pharmacological inhibitor ZDON. Mucociliary clearance was evaluated by video microscopy using fluorescent microspheres.

[0068] Results: TG2 expression was upregulated in a sub-group of asthma patients and upon IL-13-mediated hypersecretory differentiation and correlated with MUC5AC expression (FIGS. 1A-1B). TG2-high epithelia showed decreased microsphere transport. Indeed, TG2 upregulation is observed in in vitro regenerated bronchial epithelia in asthma and is associated with poorer mucociliary clearance (FIGS. 2A-2B). TG2 colocalized with MUC5AC at the surface of the bronchial epithelium in asthma. TG2 expression levels are upregulated in ALI-regenerated epithelia from cystic fibrosis and asthma patients and correlate with TGF-beta2 expression levels (FIGS. 3A-3C). TG2-exposed respiratory mucins incorporated the biotinylated amine BAP (FIG. 4). IL-13 promoted MU5AC tethering to in vitro reconstituted hypersecretory epithelium (FIGS. 5A-5D), and this was blocked by ZDON. Indeed, the TG2 inhibitor ZDON tends to ameliorate mucociliary clearance in hypersecretory epithelia (FIGS. 6A-6B).

[0069] Conclusion: TG2 would participate in respiratory mucin modifications in asthma, and contribute to mucus tethering to the airway wall.

REFERENCES

[0070] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.